Our goal is to understand control of protein folding. Cellular stress proteins such as cpn60 (a 60 kDa chaperonin ) can prevent misfolding by binding intermediates. Cpn's respond to disease, and they are used diagnostically, e.g. breast cancer. This proposal focuses on molecular details of cpn60 function when it interacts with the enzyme, rhodanese. A major simplification is possible because cpn6O, alone, displays essential cpn functions. Rhodanese is ideal, because: it is monomeric; we have mutants and stabilized intermediates; and it can be refolded spontaneously, or with cpn60, or, analogously, with detergents. We focus on the questions: How can cpn60 distinguish native from partially-folded proteins? What is the role of cpn60 quaternary structure? How is cpn60 binding modulated? We speculate that induced conformational changes in cpn60 alter hydrophobic contacts which, along with ionic interactions, are central to the cpn60 mechanism. The Specific Aims are designed to extend ongoing research and test five hypotheses: I. Hydrophobic regions on cpn60 can be modulated by nucleotide binding and ionic interactions. We will study hydrophobic exposure with fluorescent probes, and monitor effects of nucleotides, ions, pH and peptides. II. Specific interacting regions on proteins and on cpn60 can be identified. We will use: synthetic peptides representing stabilized amphiphilic helices; cross linking; and bisANS photo incorporation to identify cpn60 binding sites. Disulfide formation will test for cpn60 helix induction in peptides. III. Changes in protein flexibility and changes in subunit interactions are vital to cpn60 function For flexibility, we will use fluorescence and tritium exchange; and for overall size, we will use ultracentrifugation, high precision gel filtration, and light scattering. Urea perturbation will probe stability of quaternary structure. IV. Cpn60 monomers possess essential functions of the holochaperonin. Low [urea] or high pressure will produce monomers. Emphasis will be on hydrophobic exposure and flexibility in response to binding ligands. V. The conformational stability of folding intermediates can influence interactions with cpn60. We will study: a) cpn60 binding of rhodanese mutants; b) requirements for cpnl0 and ATP; and c) heterogeneity of bound intermediates.